Extract of Euodia suaveolens Scheff, repellent compositions and use thereof

ABSTRACT

The invention relates to an extract of Euodia suaveolens Scheff. The invention is characterized in that the extract is obtained from the airborne portions of the plant by means of at least one extraction step using an organic or hydro-alcoholic solvent, and in that it comprises evodone at a concentration of between 0.1 and 35% by weight of the total weight of the extract, and in that it has an insect repellent activity. The invention also relates to a repellent composition, characterized in that it comprises, in a cosmetically acceptable medium, evodone at a concentration of between 0.1 and 25% by weight of the total weight of the composition, and to the use of evodone as an active agent for repelling insects, in particular mosquitoes and more particularly the European/Mediterranean mosquitoes of the Aedes aegypti species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/912,640 filed Jun. 7, 2013, which is a divisional of U.S. applicationSer. No. 13/256,078 filed Sep. 12, 2011, which is a U.S. national stageof PCT/FR2010/000209 filed Mar. 12, 2010, the contents of each of whichare hereby incorporated by reference herein in their entirety.

The present invention relates to an extract obtained from airborneportions of the plant Euodia suaveolens Scheff, repellent compositions,as well as their use for repelling insects, in particular mosquitoes andmore particularly the European-Mediterranean mosquitoes of the Aedesaegypti species.

Environmental, climatic and societal changes in Europe and countries ofthe South influence the distribution and dynamics of pathogens and theirvectors, thus contributing to the emergence/re-emergence of diseasessuch as malaria, dengue fever or Nile virus, with considerable economicand health impacts.

The fight against vectors is the best large-scale prevention strategyapplicable to most tropical diseases transmitted by insects, especiallythose transmitted by mosquitoes. Indeed, for some diseases such asmalaria, yellow fever, encephalitis, dengue fever, Chagas disease and,more recently, chikungunya, there is no immediately effective vaccine orcurative means.

Thus, the strategies used to reduce the incidence of vector-bornediseases have focused on the elimination or control of vectorpopulations by chemical and biological means, such as insecticides, andon personal protections, primarily in the form of repellent products.The latter are notably subject to renewed interest in the field ofpublic health. The use of repellents can also become an effectivepersonal protective measure in those areas where the biology andbehavior of vectors make the use of conventional methods such asimpregnated materials, or the spraying of insecticides, lessappropriate, such as in South East Asia or in the Indian Ocean.

However, insecticides are known to be toxic and can irritate therespiratory tract, eyes and skin, or cause nervous system orgastrointestinal disorders, or also accumulate in the fatty tissue ofhumans.

Therefore, it is preferred to use repellents, which are synthetic ornatural chemicals, capable of causing negative chemotropism in pests.Repellents are also known as insectifuges or “mosquito-repellents”. Theydo not generally kill the insects, but keep them away. Repellents alterthe insect's orientation ability, thus diverting it from its potentialtarget.

Many stimuli (visual, thermal, olfactory) are involved when insectslocate their prey. Thus, to date, the exact mechanism acting inrepellents is not yet known. In addition, few efficacy and long-termtoxicity studies have been performed on repellents.

Essential oils are generally known to have a lower repellent power thansynthetic products.

The efficacy of a repellent depends on its concentration, the targetinsect, the excipient (volatility, extended-release, . . . ), but alsoon many other environmental factors.

Chemical repellents include, in particular, products based on DEET(N,N-diethyl-3-methylbenzamide or N,N-diethyl-m-toluamide) with thefollowing chemical structure:

This synthetic compound is known as the most commonly used and mosteffective repellent on the market. Approximately 200 million people useDEET each year and more than 8 billion doses have been applied over thelast 50 years. It has been shown that DEET blocks theelectrophysiological response of the insect's sensory neurons, which areinvolved in olfactory attraction. It confers a protection periodgenerally ranging from 2 to 8 hours depending on the compounds withwhich it may be associated in the products, and more specificallydepending on its concentration. For example, it confers a protectionperiod of more than 5 hours for DEET at a dose of 23.8 percent,according to a study carried out in the laboratory on Aedes aegypti(Fradin M S, Day J F “Comparative efficacy of insect repellents againstmosquito bites”, New England Journal of Medicine, 2002).

However, DEET has an unpleasant and persistent odor, a greasy texture onthe skin, is irritating to the eyes and mucous membranes, and also hasan aggressive effect on certain plastics (watches, glasses). Thus, eventhough it has been re-registered by the Environmental Protection Agency,it can be toxic at a concentration greater than 30%, especially inchildren under the age of 12, pregnant women and nursing mothers. Inaddition, its toxicity being related to skin permeability, since 30% ofa dose of DEET is absorbed through the skin, and to its storage capacityin the skin, since urinary excretion represents 10 to 15% of its dose,it may be advisable not to use it at a concentration greater than 30%.

However, a concentration of DEET below 30% has little efficacy againstmosquitoes in individuals over 12 years of age, as its activeconcentration is 30 to 50%.

Recently, studies have shown that DEET might increase the concentrationof acetylcholine in the synaptic cleft by inhibitingacetylcholinesterase (Vincent Corbel et al. “Evidence for inhibition ofcholinesterases in insect and mammalian nervous system by the insectrepellent DEET”. BMC Biology, August 2009). Acetylcholinesterase is akey enzyme in the transmission of nerve impulses. Once information hasbeen transmitted, the chemical mediator, acetylcholine, degrades.Inhibition of acetylcholinesterase leads to an accumulation ofacetylcholine, which maintains permanent transmission of nerve impulses,which may lead to muscle tetany.

Although serious side effects nevertheless remain exceptional and areoften linked to an overdose or overuse, this discovery once again posesthe issue of safety in the use of DEET and raises the hypothesis thatDEET represents a risk of nervous toxicity in the human species.

Also known is icaridin or KBR3023 or 1-methylpropylester, which is asynthetic chemical repellent, having the following chemical structure:

It is described as having an efficacy comparable to that of DEET, but atlower concentrations (19.2% icaridin vs. 30% DEET). Icaridin is almostodorless, does not cause skin irritation, although it produces only aslight irritation of the eye, and has no adverse effect on plastics. Theuse of icaridin in effective amounts of between about 20 and 30% ishowever not recommended in children under 30 months and pregnant women.

Synthetic repellents available on the market also include IR3535 orethyl-butyl-acetyl-aminopropionate, having the following chemicalstructure:

The active concentration of IR3535 is between 20 and 35%. It is the onlychemical repellent not prohibited for pregnant women. Its acute andchronic toxicities are low. According to the EPA, on the basis of itsuse for 20 years in Europe with no significant adverse effect, the riskis considered to be minimal to nil. However, its effectiveness is foundto be lower than that of DEET, with an average efficacy of 4 hrs forIR35/35 above 20%.

There are other known repellents, such as citriodiol or p menthane 3.8diol (a natural Eucalyptus extract), neem, or geraniol, which arenatural extracts available on the market. These prove to be effectiveand can provide protection which generally has a maximum persistence of6 hours for citriodiol, to 2 hours.

Other natural extracts such as extracts of Spilanthes (Acmella oleracea)or Sichuan pepper (Zanthoxylum piperitum), may also prove to beeffective as repellents. However, the active ingredients of Spilanthesand Sichuan pepper are alkylamides, which may be skin irritants atconcentrations of the order of 15 to 20%.

Natural repellents also include oils and essential essences, extractedby steam distillation and expression, in particular those of geranium,garlic, lavender, or also citronella which, however, are not consideredto be very effective, and provide protection with a persistence of about2 hours to 30 minutes, or even less, depending on the wind and outsidetemperature. They require much more frequent applications than syntheticrepellents to maintain an effective level of protection. They can alsobe too odorous. Furthermore, the pure essential oil of Euodia suaveolensScheff appears, according to tests conducted by the applicant, to beeffective but highly volatile and above all highly odorous and thereforenot usable as such.

In general, there is a real demand for the provision of products orpreparations, which would advantageously be of natural origin, much lessharmful to human health, and biodegradable. In the present context,particularly in the context of biocide regulation, there is a need tofind a natural or synthetic repellent, which would be less toxic andless irritating, at least as effective as DEET and icaridin, and wouldnot have the adverse effects of prior art repellents.

Euodia suaveolens Scheff is a plant of the Rutaceae family, better knownas Zodia, or also Tanaman Zodia. Approximately 150 genera of Euodia, andperhaps more than 1500 species, are described in the literature, amongwhich Euodia suaveolens Scheff, although it is referred to as Evodia inthe scientific literature dealing with this genus. This Indonesian plantoriginates from the region of Irian, in Western Papua New Guinea, whereit is known under local names such as Halau nyamuk, Durian Hantu Hutanor Sukang or Sungei. It is also found on the island of Java and otherAsian countries. Euodia suaveolens Scheff is a bushy evergreen shrubthat can reach a height of between 0.3 and 3 meters, in general 0.75meters. Its thin leaves, of a light green color and with a shiny, waxyand smooth aspect, are simple and linear, long-stalked and may reach alength of 20-30 cm for mature foliage. Their central vein protrudes fromthe underside. They have a bitter taste. Their flowers are pale yellow,arranged in clusters and flourish in the summer. This plant gives off astrong aromatic odor.

Inhabitants of areas where this plant is grown have used Euodiasuaveolens Scheff for many years in traditional medicine. In particular,its leaves are used to fight malaria and other diseases vectorized bymosquitoes, such as dengue. It also appears that wounds resulting fromattacks by infected mosquitoes heal faster when placed in contact withthe foliage of this plant.

The repellent activity of the leaves of Euodia suaveolens, after theskin has been rubbed with them, against dengue carrying mosquitoes, hasalso been reported (Agus Kardinan, 2005, Services Laboratory, SeameoBiotrop, Zodia—Mosquito repellent plant). The gas chromatographyanalysis of the essential oil obtained from leaves of this plant,conducted by the Balai Penelitian Tanaman Rempah dan Obat (Baittro)revealed the presence of 46% of linalool, having the following chemicalstructure:

and 13.26% of alpha-pinene having the following chemical structure:

which are molecules considered to be repellents by some researchers. Itis also reported in the literature that, in the opinion of otherresearchers, this repellent activity could be due to evodiamine, havingthe following chemical structure:

and rutaecarpine, having the following chemical structure:

which are present in the leaves of Euodia suaveolens.

In spite of these teachings, it should be noted that there has not beenany report, or even suggestion, concerning a repellent that would beeffective against insects, in particular mosquitoes, from extracts ofEuodia suaveolens Scheff, in particular those not containing aneffective amount of linalool, alpha-pinene, or evodiamine or alsorutaecarpine.

In consideration of the above, a problem addressed by the invention isthat of developing a natural or synthetic repellent, capable ofrepelling insects as effectively as a known synthetic chemical insectrepellent, and which does not have the side effects observed in theprior art.

Thus, with a view to meeting this need, the applicant has unexpectedlydiscovered that extracts derived from Euodia suaveolens Scheff that donot contain linalool, alpha-pinene, evodiamine and/or rutaecarpine or,at least not in an effective amount, show remarkable repelling activityagainst insects, in particular mosquitoes and, particularly,European/Mediterranean mosquitoes of the Aedes aegypti species, thusproviding a new response in terms of protection against insects, whileat the same time preserving the environment and human health. Theapplicant has also specifically identified the active agent responsiblefor such a repellent activity.

The extract and repellent agent according to the invention offer thefollowing advantages over the prior art:

-   -   the extract is natural;    -   the extract and active agent have a more efficient repellent        activity against insects, in particular against mosquitoes and        particularly against European/Mediterranean mosquitoes of the        Aedes aegypti species, than prior art natural extracts;    -   the extract and active agent, depending on their concentration,        have a repellent activity which is as effective as chemical        repellents and more particularly DEET;    -   the active agent can be used at a low dose; and    -   the extract and compositions comprising said active agent do not        have the adverse effects of chemical insecticides and insect        repellents.

A first object of the invention is therefore to provide an extract ofEuodia suaveolens Scheff, characterized in that it is obtained from theairborne portions of the plant by means of at least one extraction stepwith an organic or hydro alcoholic solvent and in that it comprisesevodone at a concentration of between 0.1 and 35% by weight of the totalweight of the extract, and in that it has an insect repellent activity.

In addition, a second object of the solution of the invention is toprovide a repellent composition, characterized in that it comprises, ina cosmetically acceptable medium, evodone at a concentration of between0.1 and 25% by weight of the total weight of the composition.

A third object the invention is also to use evodone as an active agentfor repelling insects, in particular mosquitoes and more particularlyEuropean/Mediterranean mosquitoes of the Aedes aegypti species.

The invention and benefits derived therefrom will be better understoodafter reading the following non-limiting description and embodimentswith reference to the accompanying figures, in which:

FIG. 1 is a 3D chromatogram obtained by high-performance thin layerchromatography (HPTLC) at 280 nm using a scanner, revealing the presenceof evodone in different extracts of Euodia suaveolens Scheff;

FIG. 2 is a reading at 254 nm before the development of a HPTLCchromatogram, showing different extracts of Euodia suaveolens Scheff,revealing the absence, or simply the presence of trace amounts, ofrutaecarpine and evodiamine in the extracts according to the invention;

FIG. 3 is a chromatographic profile obtained by gas chromatography withflame ionization detection on a polar column, which characterizes atleast the partial composition of an extract of Euodia suaveolens Scheffaccording to the invention;

FIG. 4 is a reading of a HPTLC chromatogram after visible lightdevelopment, showing different extracts of Euodia suaveolens Scheff andrevealing the presence of evodone in the extracts according to theinvention; and

FIG. 5 is a reading of a HPTLC chromatogram after visible lightdevelopment, showing different extracts of Euodia suaveolens Scheff andrevealing the presence of evodone in the extracts according to theinvention.

In this description, unless otherwise specified, it is understood thatwhen a concentration range is given, it includes the upper and lowerbounds of said range, and that the concentration is given by weightrelative to the total weight of the composition or extract concerned.

The extract of Euodia suaveolens Scheff according to the invention isisolated from the airborne portions of the plant.

By airborne portions of the plant is meant the stems, leaves and flowersof the plant. These can be used fresh or dried, advantageously dried.

To obtain the extract according to the invention, the airborne portionsof Euodia suaveolens Scheff are advantageously harvested, dried andcoarsely ground.

At least one extraction step is then performed using organic orhydro-alcoholic solvents. This extraction step is different from anextraction step for the production of an essential oil or essence, whichis obtained strictly by steam distillation or expression.

The extraction is advantageously repeated several times, for example 3times.

It is performed with a plant/extraction solvent ratio, by weight/volume,between about 1/1 and about 1/20, for example 1/5 or 1/10.

According to the invention, the temperature of the extraction solvent isgreater than or equal to about 15° C., or room temperature.Advantageously, the maximum extraction temperature is the refluxtemperature of the extraction solvent. For example, the extraction iscarried out at 75° C. with ethanol.

According to the invention, the extraction is advantageously dynamic andis performed under stirring.

According to the invention, the extraction time is between 30 minutesand 5 hours for each extraction step, for example 3 or 4 hours.

Non-limiting examples of organic solvents of the invention includealkanes, ether-oxides, esters, alcohols, halogenated hydrocarbons,carbon dioxide in the supercritical state.

The organic solvent according to the invention is preferably selectedfrom pentane, hexane, heptane, octane, cyclohexane, tetrahydrofuran,dioxane, diethyl ether, ethyl acetate, isopropyl acetate, methanol,ethanol, propanol, isopropanol, butanol, methyl ethyl ketone, dimethylketone, methyl isobutyl ketone, dichloromethane, dimethyl carbonate orcarbon dioxide in the supercritical state or a mixture of theabove-mentioned solvents, preferably hexane and ethanol.

Advantageously, the organic solvent is selected from organicallyproduced solvents, for example alcohols and carbon dioxide in thesupercritical state.

According to the invention, the hydro-alcoholic solvent, for example, isan ethanol/water dilution solvent with a ratio of 80/20 by volume.

In the preparation of an extract according to the invention, aseparation of the solid phase from the liquid phase is then carried out.The solid portions of the plant are thus separated from the liquidextraction juice by plate filtration or centrifugation, pressing.

The thus obtained filtrate is then concentrated by evaporation, to yielda more or less concentrated extract, if appropriate until totalevaporation of the extraction solvent.

Advantageously, the filtrate can be concentrated by vacuum evaporation.

The dark green-brown concentrated extract can then be bleached orrefined using activated carbon, or acidic or clayey earths, or moleculardistillation or extraction with carbon dioxide in the supercriticalstate or codistillation.

The extract according to the invention can then be purified. It can bepurified by any technique known to one skilled in the art, such as, forexample, liquid phase-liquid phase extraction between 2 immisciblesolvents, absorption onto a carrier such as silicate or an ion exchangeresin, precipitation or crystallization.

The mass yield of the extract is between about 1% and about 25% of thedry matter involved, for example about 15%.

According to the invention, different types of extractions can beperformed, such as maceration (static extraction at room temperature),hot static extraction, decoction, leaching.

According to an advantageous embodiment of the invention, from aquantitative point of view in terms of yield, a Soxhlet extraction ordynamic extraction, maceration, static hot extraction, ultrasonicextraction or microwave extraction with ethanol at a concentrationgreater than about 80%, for example 80%, 96% or 97%, is carried out.

After purification, the extract according to the invention can be driedby total evaporation of the solvent and then dissolution in a compatibleand cosmetically acceptable solvent.

Another feature of the extract is that it does not contain linalool,alpha-pinene, or evodiamine, rutaecarpine, or at least not in aneffective amount according to the prior art. Even though these compoundsare present in the plant, the extract or oleoresins obtained from thisplant contain only a small amount of linalool and alpha-pinene andneither evodiamine nor rutaecarpine.

Furthermore, the extract according the invention has a zero or verysmall titer of these molecules, for example a content of less than 1%,preferably less than 0.1%, and therefore contains only trace amounts ofthese molecules. Indeed, linalool and alpha-pinene are removed, inparticular, during the organic or hydro-alcoholic extraction of theextract according to the invention. These molecules are driven by thedistillation of the solvent and are diluted by concentrating theextract. For evodiamine and rutaecarpine, the solvent used for theextraction according to the invention is not selective for these typesof alkaloid structures. The extract according to the invention, whentested on European/Mediterranean mosquitoes of the Aedes aegyptispecies, therefore no longer contains, or contains only trace amounts ofthese four molecules. According to the invention, the propertiesdisplayed by such an extract are not attributable to the presence ofsuch substances.

The extract according to the invention, which, in itself, is a newproduct, however contains non-volatile compounds such as flavonoids, forexample glycosylated flavanols such as rutine or 3 O-rutinosylquercetol,flavonols such as kaempferol and isorhamnetin or methyl-3-quercetin, aflavone, such as a derivative of apigenine, for example vitexin orvitexin 2″O rhamnoside, as well as volatile compounds such as bicycliclactones, in particular evodone or (6S)3-6 dimethyl 6,7 dihydro 5Hbenzofuran-4-one, menthalactone or mintfuranone or3,6-dimethyl-5,6,7,7a-tetrahydro-4H-1-benzofuran-2-one, aromacurcumene,and fatty acids, in particular of the acidic, palmitic or oleic type.

In particular, the extract of the invention contains a titer of evodone,having the following chemical structure:

Evodone is an oxygenated monoterpene, that is, a benzofuranone derivedfrom menthofuran. Its description can be found, in particular, in Euodiahortensis (Rutaceae). The plant Calamintha ashei (Lamiaceae) alsocontains oxygenated monoterpenes such as evodone, caryophyllene,menthofuran, and calaminthone. These compounds have been studied fortheir allelopathic properties for the inhibition of the seeds of certainspecies such as Lactuca sativa and Schizachyrium scoparium. A mixture ofthese terpenes appears to inhibit seed germination, but the individualcompounds do not seem to have a significant activity.

The synthesis of evodone can be performed from5-methylcyclohexan-1,3-dione in the presence of chloroacetone (A.Srikrisma and G. Veera Raghava Sharma, “A simple synthesis(+/−)evodone”, Indian journal of Chemistry, 1989, p. 852). 99% pure evodone(racemic mixture) was thus obtained.

The evodone titer of the extract according to the inventionadvantageously corresponds to a concentration of between about 0.1 and35% by weight of the total weight of the extract, preferably between 0.2and 5%.

For example, a crude, unrefined oleoresin according to the invention maycomprise between 0.1 and 35% of evodone, for example 0.8% for anunrefined crude extract, and 20.16% when the codistilled oleoresin istaken up in hexane, and a codistilled refined extract may includebetween 0.1 and 20% of evodone, for example 2%.

As shown in FIGS. 1, and 3 to 5, evodone is present in an extract ofEuodia suaveolens Scheff according to the invention.

In FIG. 1, chromatograms 3 to 14 represent the deposited evodonestandard at the same concentration and different volumes, and arecharacterized by the presence of a peak having a baseline (front ratio)of 0.69 for this mobile phase in this particular analysis case. It isobserved that the same evodone-characteristic peak is present inchromatograms 1, 2, 15, 16, 17 pertaining to extracts of Euodiasuaveolens Scheff, namely, respectively, in an oleoresin obtained fromfresh leaves, an oleoresin obtained from dry leaves, a formulation basedon a codistilled extract with an evodone content of 0.3%, anotherformulation based on a codistilled extract with an evodone content of2.36%, and codistilled oleoresin taken up in hexane.

Furthermore, analysis of the essential oil of Euodia suaveolens Scheff,by comparing the essential oil with the oleoresin extracted according tothe invention, reveals various compounds identified by gaschromatography coupled with mass spectrometry.

The Table below shows some of the compounds mentioned above anddescribed by some authors as being responsible for the repellentactivity (including alpha pinene and linalool). This Table shows that,based on the obtained results, alpha pinene and linalool, in particular,are not present in the oleoresin extracted according to the invention.However, the oleoresin extracted according to the invention retains astrong repellent activity against mosquitoes.

Content of compounds Theoretical content analyzed GC/FID of compounds inthe oleoresin content in in the oleoresin extracted according theanalyzed extracted according to the invention, Compounds essential oilto the invention in GC/FID alpha pinene 0.12% 0.0086%  not detectedLimonene 18.77% 1.13% not detected Linalool 0.08% 0.00480%   notdetected Menthofuran 31.30% 0.28% not detected Limonen 10 ol 6.21% 0.38%2.28%

For information, the theoretical levels given in the Table have beenextrapolated from analyses of the essential oil. Based on this data, itis indeed possible to determine the theoretical contents of thesecompounds in the plant, and then, in the oleoresin extracted accordingto the invention.

However, the fact that the oleoresin according to the invention isobtained by alcoholic extraction followed by concentration, should betaken into account. Thus, when the solvent is evaporated, most of thesemolecules, which are volatile molecules, are primarily driven byazeotropy, which explains why they are not found in the extractaccording to the invention.

In addition, as shown in FIG. 2, rutaecarpine and evodiamine are notpresent in the extract of the invention, or are present in trace amountsonly.

Specifically, in FIG. 2, different extracts of Euodia suaveolens Scheffhave been analyzed: lane 1 relates to a concrete obtained by hexaneextraction from fresh Euodia suaveolens Scheff leaves; lane 2 relates toan absolute obtained by alcohol extraction of the concrete; lane 3relates to a bleached oleoresin according to the invention; lane 4relates to an extract according to the invention, bleached by moleculardistillation; lane 5 relates to an essential oil of Euodia suaveolensScheff; lane 6 is empty; lane 7 corresponds to the evodiamine standardwith a baseline (retardation ratio) of 0.35-0.39, shown to be 0.38 inFIG. 2, under these analytical conditions; lane 8 corresponds to therutaecarpine standard with a baseline (retardation ratio) of 0.53 underthese analytical conditions; lane 9 is empty; and lane 10 corresponds tothe evodone standard.

In lanes 3 and 4, the absence of a band with a baseline of 0.53(retardation ratio) is observed, which is characteristic of the presenceof rutaecarpine in these extracts. Thus, the extracts according to theinvention deposited in lanes 3 and 4 are free of rutaecarpine. It isalso observed in lanes 3 and 4 that there is no significant band havinga baseline of 0.38 (retardation ratio), which is characteristic of thepresence of evodiamine in these extracts. Only trace amounts aredistinguished, which might indicate the presence of traces of evodiaminein the extracts according to the invention.

Furthermore, as shown in FIG. 3, the composition of a bleached ethanolextract of Euodia suaveolens Scheff according to the invention has beencharacterized at least partially by GC/FID chromatography (AgilentTechnologies HP 6890) on a polar column (Varian CP8713 CP WAX 52 CB; 50m×0.25 mm×0.2 mm) following a rise in temperature of 70° C. for 5 min,then of 2° C./min up to 240° C. for 50 min, and for an injected volumeof 1 μl. In this extract, peaks that are characteristic of the presenceof benzaldehyde (peak 1), carbitol (peak 2), aroma curcumene (peak 3),benzyl alcohol (peak 4), evodone (peak 5), limonene-10-ol or(p-mentha-1,8(10)-dien-10-ol) (peak 6), levojunenol (peak 7),menthalactone isomer 1 (peak 9), menthalactone isomer 2 (peak 10),phytol (peak 12), and peaks that are characteristic of the presence offatty acids, can be observed.

As illustrated in FIG. 4, HPTLC analyses of evodone and various extractsof Euodia suaveolens Scheff according to the invention were carried out:lane 1 relates to an extract of Euodia suaveolens Scheff obtained bycodistillation on monopropylene glycol in bleached oleoresin; lane 2relates to an extract obtained by codistillation on monopropylene glycolof a non-bleached oleoresin obtained from fresh Euodia suaveolens Scheffleaves; lanes 3 to 7 correspond to the evodone standard; lane 8 relatesto a crude unrefined oleoresin obtained by ethanol extraction from freshEuodia suaveolens Scheff leaves; lanes 9 and 10 correspond to theevodone standard; lane 11 relates to a bleached oleoresin obtained byethanol extraction from dry leaves of Euodia suaveolens Scheff; lane 12relates to a bleached oleoresin obtained by ethanol extraction fromfresh leaves of Euodia suaveolens Scheff; lane 13 is empty; lane 14corresponds to the evodone standard.

Thus, the extracts according to the invention that are deposited inlanes 1, 2, 8, 11 and 12 do contain evodone.

As shown in FIG. 5, HPTLC analyses of evodone and various extracts ofEuodia suaveolens Scheff according to the invention were carried out:lane 1 relates to an unrefined crude oleoresin obtained by ethanolextraction from dry leaves of Euodia suaveolens Scheff; lane 2 relatesto an unrefined crude oleoresin obtained by ethanol extraction fromfresh leaves of Euodia suaveolens Scheff; lanes 3 to 14 correspond tothe evodone standard; lanes 15 and 16 relate to formulations obtainedfrom codistilled oleoresin extracts; lane 17 is empty; lane 18 relatesto hexane extraction of a codistilled oleoresin.

Thus, the extracts of the invention deposited on lanes 1, 2, 15, 16, 18do contain evodone.

The extract according to the invention is natural. Advantageously, theextract can be certified as organic, using organic extraction solventssuch as alcohols or carbon dioxide in the supercritical state.

The extract is biodegradable and non-toxic. Indeed, a toxicity study ofan ethanol extract of Euodia suaveolens Scheff was performed at aconcentration of 100% pure, non-bleached, on six female Sprague Dawleyrats, aged 8 weeks and weighing between 186 g and 210 g. The study beganafter an acclimatization period of 5 days. All factors such asbrightness, temperature and humidity were controlled. The results areobtained by comparison with a control group of rats that receiveddimethyl sulfoxide only. Each rat received a single effective dose of2000 mg/kg by body weight of the pure extract of Euodia suaveolensScheff, diluted in dimethyl sulfoxide and administered orally, inparticular by force-feeding, at a volume of 5 ml/kg of body weight.Based on the results obtained, the extract according to the invention isnot toxic. Specifically, it has an LD50 of more than 2000 mg/kg of bodyweight. According to the OECD 423 directive, the LD50 can be consideredto be greater than 5000 mg/kg by bodyweight after oral administration inrats.

As an example of a method for extracting and obtaining an extract ofEuodia suaveolens Scheff or oleoresin according to the invention, thesteps described below, in particular, are carried out.

The previously dried leaves and flowers of Euodia suaveolens Scheff arefirst coarsely ground.

600 g of non-finely ground plant are thus added to a 20 L three-neckedflask placed in a water bath fed with steam.

The extraction is performed with 96% ethanol. Three extractions areperformed.

The first extraction is performed with a volume of 10 times the weightof the ground plant, or 6 L. The extraction is carried out under refluxwith mechanical stirring for 3 hours, at a temperature of 75° C.

The filtrate is then discharged and collected.

A second extraction is then performed with a volume equal to 6 times themass of the initial plant, or 3.6 L, under reflux, with stirring for 3hours.

Thereafter, a final extraction is performed with a volume equal to 4times the initial mass of plant, that is 2.4 L.

The three filtrates are combined and then filtered on a Büchner filter.

A clear, dark green extract is obtained.

The extract according to the invention is then concentrated under vacuumusing a rotary evaporator at a temperature of 60° C.

An oleoresin according to the invention, dark green in color, extractedfrom Euodia suaveolens Scheff, is thus obtained. It has a pastyappearance, is solid at room temperature and viscous-liquid when hot.

The extract according to the invention is then bleached with activatedcarbon at a content of 10% by weight in an 80/20 ethanol/water mixture.The finally obtained extract according to the invention isreddish/brown, glossy, and soluble in an 80/20 ethanol/water mixture.

The extract according to the invention is moderately fragrant.

Alternatively, the extract according to the present invention isbleached with supercritical CO₂.

Various tests using supercritical CO2 were carried out on the aboveoleoresin, for example at a pressure of 180 bar and at a temperature of45° C., or at a pressure of 250 bar and at a temperature of 45° C., with10% ethanol as co-solvent.

The CO₂ test without a co-solvent results in a lemon yellow, odorousextract, but having a non-uniform appearance.

The CO₂ extract obtained with an ethanol-based co-solvent allows areddish brown, less odorous extract to be obtained.

According to a preferred embodiment, the oleoresin obtained from freshplants is codistilled on a cosmetically acceptable carrier such asmonopropylene glycol. This codistillation provides a pale yellow extracthaving a high concentration of evodone, for example about 2%.

This extract can then be taken up in hexane to increase the evodonetiter.

According to a second aspect, the invention also pertains to a repellentcomposition comprising, in a cosmetically acceptable medium, evodone ata concentration between 0.1 and 25% by weight of the total weight of thecomposition.

Preferably, the repellent composition according to the inventioncomprises evodone at a concentration of between 0.2 and 10% by weight ofthe total weight of the composition, for example at a concentration of3% by weight of the total weight of the composition.

The invention also relates to a composition comprising an effectiveamount of an extract of Euodia suaveolens Scheff as defined above, asthe active repellent agent against insects, in particular againstmosquitoes. It is incorporated into the composition with anycosmetically acceptable excipient. Such a composition is substantiallyfree of any effective amount of linalool, alpha-pinene, evodiamine orrutaecarpine, and, in particular, has an evodone titer, at aconcentration of between 0.1 and 25% by weight of the total weight ofthe composition.

Advantageously, the extract content in the above composition is between1 and about 70% by weight of the total weight of the composition,preferably 10.10%, 26.8%, 32% or 35%.

For example, a composition according to the invention may comprise 35%of an ethanol extract (ethanol/water as dilution solvent: 80/20), 10.10%of an extract obtained by codistillation with monopropylene glycol, or26.8% of an extract obtained by alcoholic extraction, formulated withcastor oil and ethanol.

The compositions according to the invention may be more or less fluidand may be in any pharmaceutical form suitable for use in a topicalapplication to the skin, for example, in the form of a milk, gel, cream,lotion, emulsion, spray and preferably in the form of a spray.

Examples of solvents used in the composition according to the inventioninclude: water, ethanol, glycerin, propylene glycol, sorbitol.

Said composition may optionally comprise another active ingredientselected from agents which have a recognized repellent effect oninsects, preferably a naturally occurring repellent agent, for examplementhoglycol or citriodiol.

This naturally occurring agent is derived from the essential oil ofEucalyptus citriodora.

By way of illustration, some formulation examples of compositionsaccording to the invention are mentioned below, which contain evodone orthe above-mentioned extract of Euodia suaveolens Scheff:

EXAMPLE 1 Spray Against European/Mediterranean Mosquitoes (Aedesaegypti) for Topical Application

32% of extract of Euodia suaveolens Scheff

1.3% of citriodiol

65% of 80%-alcohol

1.7% of monopropylene glycol

EXAMPLE 2 Spray Against European/Mediterranean Mosquitoes (Aedesaegypti) for Topical Application

3% of evodone

63% of 100%-alcohol

34% of monopropylene glycol

EXAMPLE 3 Spray Against European/Mediterranean Mosquitoes (Aedesaegypti) for Topical Application

26.8% of extract of Euodia suaveolens Scheff

33.5% of castor oil

39.7% of 100%-alcohol

In a third aspect, the present invention relates to the use of evodoneas an active agent for repelling insects, particularly mosquitoes andmore particularly European/Mediterranean mosquitoes of the Aedes aegyptispecies.

The invention also relates to the use of an extract of Euodia suaveolensScheff as an active agent, or of compositions as defined above to repelinsects, in particular mosquitoes and more particularlyEuropean/Mediterranean mosquitoes of the Aedes aegypti species.

In this respect, the invention is illustrated by the following efficacytests, carried out in the laboratory, where the formulas are notdegraded through the effects of abrasion, exudation, sunlight,temperature or even swimming in the sea. These tests are designed todemonstrate the repellent effect of evodone and an extract of Euodiasuaveolens Scheff at different concentrations, purified, whetherbleached or not, alone or in combination with another natural activerepellent agent such as menthoglycol, on European/Mediterraneanmosquitoes of the Aedes aegypti species.

The tests are carried out on female Aedes aegypti mosquitoes aged 6 to10 days having undergone a prior 24-hour period of fasting (no bloodmeal) in order to increase their aggressiveness.

The blood meal target is a mouse of the “hairless” type held in aretaining cage (using eye protection) and coated with the test product(0.1g/60 cm²) applied with a pipette.

30 minutes after product application, the mouse is introduced into acage containing approximately 200 female mosquitoes (prior removal ofthe males using a food gradient).

The test lasts 5 minutes and during this period the number of mosquitoeshaving landed on the mouse (“landing” effect: number of mosquitoes thatland on the treated area and leave without biting) and the number ofeffective bites, are recorded.

The test is stopped after 5 effective bites (“stop” in the Tablesbelow).

The test is repeated with new batches of mosquitoes, at intervals of onehour until a duration of 8 hours is reached, depending on thepersistence limit of the product (between the runs, the mouse wasreleased). Based on the outcome of the first repetition, repetitions areperformed at time intervals of 30 minutes in order to provide a moreaccurate limit for the duration of protection.

During and between the runs, the mice are maintained in a “tropical”like atmosphere under controlled conditions at 27+/−1° C. and 70+/−5%relative humidity. The cages are changed upon each unit test and placed3 meters apart.

3 repetitions are carried out per formula to be tested and on 3different mice, that is, 9 unit tests per formula.

Test 1:

4 extracts (supplier: Charabot S. A.) are tested comparatively, thesolvent serving as a reference (it is verified that at least 10 landingsoccur in 30 seconds to validate the test):

Extract 1: ethanol extract of Euodia suaveolens Scheff according to theinvention at a concentration of 35% (ethanol/water as dilution solvent:80/20) non-bleached;

Extract 2: ethanol extract of Sichuan red pepper (Soxhlet extraction) ata concentration of 19.44% (ethanol as dilution solvent);

Extract 3: ethanol extract at a concentration of 35% of menthoglycol;

Extract 4: Spilanthes extract at a concentration of 18.56% (ethanol asdilution solvent); and

Test without any product (reference): solvent.

The results are summarized in the following Table:

AEDES T0 + 2 H T0 + 4 H T0 + 6 H T0 + 8 H T0 + 10 H T0 + 12 H AEGYPTImice rep. P L P L P L P L P L P L Extract 1 1 1 0 0 0 0 0 3 stop stopstop stop stop stop 2 0 0 0 1 0 1 stop stop stop stop stop stop 3 0 0 01 0 2 stop stop stop stop stop stop 2 1 0 0 0 0 0 1 stop stop stop stopstop stop 2 0 0 0 0 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 4 stopstop stop stop stop stop 3 1 0 0 0 0 0 1 stop stop stop stop stop stop 20 0 0 1 0 2 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stopstop stop stop Extract 2 1 1 0 0 0 1 stop stop stop stop stop stop stopstop 2 0 0 0 2 stop stop stop stop stop stop stop stop 3 0 0 0 0 stopstop stop stop stop stop stop stop 2 1 0 0 0 0 stop stop stop stop stopstop stop stop 2 0 0 0 1 stop stop stop stop stop stop stop stop 3 0 0 00 stop stop stop stop stop stop stop stop 3 1 0 1 0 1 stop stop stopstop stop stop stop stop 2 0 0 0 1 stop stop stop stop stop stop stopstop 3 0 0 0 1 stop stop stop stop stop stop stop stop Extract 3 1 1 0 00 1 0 3 stop stop stop stop stop stop 2 0 0 0 1 0 2 stop stop stop stopstop stop 3 0 0 0 0 0 2 stop stop stop stop stop stop 2 1 0 0 0 0 0 0stop stop stop stop stop stop 2 0 0 0 1 0 2 stop stop stop stop stopstop 3 0 0 0 0 0 1 stop stop stop stop stop stop 3 1 0 0 0 0 0 0 stopstop stop stop stop stop 2 0 0 0 0 0 1 stop stop stop stop stop stop 3 00 0 0 0 0 stop stop stop stop stop stop Extract 4 1 1 0 0 0 0 stop stopstop stop stop stop stop stop 2 0 0 0 1 stop stop stop stop stop stopstop stop 3 0 0 0 0 stop stop stop stop stop stop stop stop 2 1 0 0 0 1stop stop stop stop stop stop stop stop 2 0 0 0 3 stop stop stop stopstop stop stop stop 3 0 0 0 1 stop stop stop stop stop stop stop stop 31 0 1 0 1 stop stop stop stop stop stop stop stop 2 0 0 0 2 stop stopstop stop stop stop stop stop 3 0 0 0 1 stop stop stop stop stop stopstop stop P: number of bites rep: repetition L: “landing”: number ofmosquitoes landing on the treated area without biting stop = the test isstopped when more than 5 bites have been observed

Under these test conditions, with the samples provided, the insectstrains and methods considered, extracts 1 and 3 showed a repellenteffect against the mosquito, with a persistence of 6 hours afterapplication.

Extracts 2 and 4, at the concentrations used, showed a repellent effectagainst the mosquito with a persistence of only 4 hours afterapplication.

Extract 1 of Euodia suaveolens Scheff, at a concentration of 35%,purified, non-bleached, thus shows a true repellent effect with apersistence of 6 hours against the Aedes aegypti mosquito.

Test 2:

3 extracts (supplier: Charabot S. A.) are tested comparatively, with thesolvent serving as a reference (it is verified that at least 10 landingsoccur in 30 seconds to validate the test):

Extract 1: ethanol extract of Euodia suaveolens Scheff according to theinvention at a concentration of 15%, bleached by molecular distillation(ethanol/water as dilution solvent: 80/20);

Extract 2: ethanol extract of Euodia suaveolens Scheff according to theinvention, bleached at a concentration of 35% (ethanol/water as dilutionsolvent: 97%);

Extract 3: menthoglycol (20%)+Euodia suaveolens Scheff (15%) mixture(ethanol/water as dilution solvent: 80/20), bleached; and

Test without any product (reference): solvent.

The results are summarized in the following Table:

AEDES T0 + 2 H T0 + 4 H T0 + 6 H T0 + 8 H T0 + 10 H T0 + 12 H AEGYPTImice rep. P L P L P L P L P L P L Extract 1 1 1 0 0 0 0 stop stop stopstop stop stop stop stop 2 0 0 0 2 stop stop stop stop stop stop stopstop 3 0 0 0 0 stop stop stop stop stop stop stop stop 2 1 0 0 0 0 stopstop stop stop stop stop stop stop 2 0 0 0 1 stop stop stop stop stopstop stop stop 3 0 0 0 0 stop Stop stop stop stop stop stop stop 3 1 0 00 0 stop Stop stop stop stop stop stop stop 2 0 0 0 2 stop Stop stopstop stop stop stop stop 3 0 0 0 0 stop Stop stop stop stop stop stopstop Extract 2 1 1 0 0 0 0 0 3 stop stop stop stop stop stop 2 0 0 0 0 02 stop stop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stopstop 2 1 0 0 0 0 0 0 stop stop stop stop stop stop 2 0 0 0 0 0 1 stopstop stop stop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop 3 10 0 0 1 0 1 stop stop stop stop stop stop 2 0 0 0 0 0 1 stop stop stopstop stop stop 3 0 0 0 1 0 2 stop stop stop stop stop stop Extract 3 1 10 0 0 0 0 1 stop stop stop stop stop stop 2 0 0 0 0 0 0 stop stop stopstop stop stop 3 0 0 0 0 0 0 stop stop stop stop stop stop 2 1 0 0 0 0 00 stop stop stop stop stop stop 2 0 0 0 0 0 1 stop stop stop stop stopstop 3 0 0 0 0 0 1 stop stop stop stop stop stop 3 1 0 0 0 0 0 0 stopstop stop stop stop stop 2 0 0 0 0 0 0 stop stop stop stop stop stop 3 00 0 0 0 0 stop stop stop stop stop stop test 1 1 stop stop stop stopstop stop stop stop stop stop stop stop without 2 stop stop stop stopstop stop stop stop stop stop stop stop product 3 stop stop stop stopstop stop stop stop stop stop stop stop 2 1 stop stop stop stop stopstop stop stop stop stop stop stop 2 stop stop stop stop stop stop stopstop stop stop stop stop 3 stop stop stop stop stop stop stop stop stopstop stop stop 3 1 stop stop stop stop stop stop stop stop stop stopstop stop 2 stop stop stop stop stop stop stop stop stop stop stop stop3 stop stop stop stop stop stop stop stop stop stop stop stop P: numberof bites rep: repetition L: “landing”: number of mosquitoes landing onthe treated area without biting stop = the test is stopped when morethan 5 bites have been observed

Under these test conditions, with the samples provided, the insectstrains and methods considered, extracts 2 and 3 showed a repellenteffect against the mosquito, with a persistence of 6 hours afterapplication.

Extract 1 showed a repellent effect against the mosquito, with apersistence of only 4 hours after application.

Extract 2 of Euodia suaveolens Scheff, at a concentration of 35%,purified, bleached, and extract 3 comprising a mixture of20%-menthoglycol and a 15%-extract of bleached Euodia suaveolens Scheff,thus show a true repellent effect, with a persistence of 6 hours againstthe Aedes aegypti mosquito.

Test 3:

A test is carried out with 4 compositions according to the invention:

Extract 1: Codistilled extract of Euodia suaveolens Scheff, formulatedat 0.3% in evodone;

Extract 2: Codistilled extract of Euodia suaveolens Scheff, formulatedat 2.36% in evodone;

Extract 3: A composition comprising synthetic evodone, formulated at 3%;

Extract 4: Oleoresin of crude, unrefined Euodia suaveolens Scheff,formulated at 0.26% in evodone.

Under these test conditions, with the samples provided, and the insectstrains and methods considered, the extracts showed a repellent effectagainst the mosquito, with the following periods of protection afterapplication:

4 hrs with Extract 1;

6 hrs with Extract 2;

6 hrs with Extract 3;

6 hrs with Extract 4.

A racemic mixture of synthetic evodone formulated at 3% (extract 3) hasa repellent activity lasting 6 hours. Evodone can be used as an activeagent to repel insects, and particularly mosquitoes. Evodone isresponsible, at least in part, for the repellent activity of the extractof Euodia suaveolens Scheff according to the invention.

Extract 2 of Euodia suaveolens Scheff comprising 2.36% evodone has amore effective repellent activity than extract 1 of Euodia suaveolensScheff comprising 0.3% evodone and obtained under the same conditions bycodistillation.

The crude, unrefined oleoresin of Euodia suaveolens Scheff, formulatedat 0.26% in evodone (extract 4), is as effective as extracts 2 and 3.Evodone is active as such and has a synergistic action in the crudeunrefined oleoresin. Evodone may act synergistically in the crudeextract according to the invention, with molecules such as otheroxygenated terpenes, for instance menthalactone, or non-polar moleculespresent in the codistilled extracts.

According to the invention, evodone has a repellent activity againstmosquitoes. A concentration of 3% or even less of synthetic evodone inextracts of crude or refined Euodia suaveolens Scheff is as effective asa concentration of 30% of DEET.

A person skilled in the art will select the cosmetically acceptablemedium so that it does not interfere with the desirable properties ofthe extract and repellent compositions according to the invention.

Of course, the invention is non limited to the embodiments and examplespresented above and a person skilled in the art, through routineoperations, may need to carry out other embodiments not exclusivelydescribed here, that broadly fall within the scope of the invention.

The invention claimed is:
 1. A method of repelling insects, comprisingapplying an effective amount of an insect-repellant compositioncomprising evodone to an area in need of being protected from insects,wherein the composition comprises the evodone at a concentration ofbetween 0.8 and 35% by weight, wherein the composition comprises atleast one of linalool, alpha-pinene, evodiamine and rutaecapine.
 2. Themethod according to claim 1, wherein the evodone is synthetic evodone.3. The method according to claim 2, wherein the evodone is synthesizedfrom 5-methylcyclohexan-1,3-dione in the presence of chloroacetone. 4.The method according to claim 1, wherein the composition comprisesevodone at a concentration of between 2% and 20% by weight of the totalweight of the composition.
 5. The method according to claim 1, whereinthe composition comprises evodone at a concentration of between 2% and10% by weight of the total weight of the composition.
 6. The methodaccording to claim 1, wherein the composition comprises evodone at aconcentration of between 2% and 5% by weight of the total weight of thecomposition.
 7. The method according to claim 1, wherein the compositioncomprises evodone at a concentration of about 3% by weight of the totalweight of the composition.
 8. The method according to claim 1, whereinthe insects are mosquitoes.
 9. The method according to claim 8, whereinthe mosquitoes are the European/Mediterranean mosquitoes of the Aedesaegypti species.
 10. The method according to claim 1, wherein thecomposition comprises at least one of linalool, alpha-pinene, evodiamineand rutaecapine at a concentration of less than 0.1% by weight of thetotal weight of the composition.
 11. The method according to claim 10,wherein the composition does not comprise any of linalool, alpha-pinene,evodiamine and rutaecapine at a concentration of more than 0.1% of thetotal weight of the composition.
 12. The method according to claim 1,wherein the composition comprises evodone at a concentration of between0.8% and 25% by weight, and wherein the composition further comprises acosmetically-acceptable vehicle selected from the group consisting ofwater, ethanol, glycerin, propylene glycol, sorbitol, alcohol, andmonopropylene glycol.
 13. The method according to claim 12, wherein thecomposition comprises evodone at a concentration of between 2% and 10%by weight of the total weight of the composition.
 14. The methodaccording to claim 13, wherein the composition comprises evodone at aconcentration of between 2% and 5% by weight of the total weight of thecomposition.
 15. The method according to claim 14, wherein thecomposition comprises evodone at a concentration of about 3% by weightof the total weight of the composition.
 16. The method according toclaim 12, wherein the insects are mosquitoes.
 17. The method accordingto claim 16, wherein the mosquitoes are the European/Mediterraneanmosquitoes of the Aedes aegypti species.
 18. The method according toclaim 1, wherein the composition is applied on at least one of clothingand human skin.
 19. The method according to claim 12, wherein thecomposition is coated by topical application on an area to be protectedfrom insects.
 20. The method according to claim 19, wherein thecomposition is coated on at least one of clothing and human skin. 21.The method according to claim 12, wherein the composition is sprayed bytopical application on an area to be protected from insects.
 22. Themethod according to claim 21, wherein the composition is sprayed on atleast one of clothing and human skin.